Indian Journal of Experimental Biology
Vol. 46, December 2008, pp. 805-810
Relaxant effects of different fractions from Nigella sativa L. on guinea pig tracheal
chains and its possible mechanism(s)
Mohammad Hossein Boskabady1, Rana Keyhanmanesh, & Mohamad Ali Ebrahimi Saadatloo2
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Received 26 March 2008; revised 8 October 2008
The relaxant effects of four cumulative concentrations of n-hexane, dichloromethane, methanol and aqueous fractions
of N. sativa (0.8, 1.2, 1.6 and 2.0 g%) in comparison with saline as negative control and four cumulative concentrations of
theophylline (0.2, 0.4, 0.6 and 0.8 mM) were examined by their relaxant effects on precontracted tracheal chains of guinea
pig by 60 mM KCl (group 1) and 10 µM methacholine (group 2). In group 1, all concentrations of only theophylline showed
significant relaxant effects. However, in group 2, all concentrations of theophylline and methanol fraction, 1.2,
1.6 and 2.0 g% concentrations of dichloromethane and 1.2 and 2.0 g% concentrations of n-hexane fractions showed
significant relaxant effects compared to that of saline. In addition, in group 1, the relaxant effect of most concentrations of
all fractions except the low concentration (0.8 g%) of dichloromethane and methanol fraction were significantly less than
those of theophylline. The relaxant effect of different concentrations of methanol and dichloromethane fraction and
1.2, 1.6 and 2.0 g% concentrations of n-hexane fraction were significantly greater in group 2 compared to group 1
experiments. There were significant positive correlations between the relaxant effects and concentrations for all fractions
(except aqueous fraction) in group 2 and for theophylline in both groups but a negative correlation for n-hexane,
dichloromethane and methanol fractions in group 1. The results showed relaxant effect of most fractions from N. sativa on
tracheal chains of guinea pigs which was more potent for methanol and dichloromethane fractions.
Keywords: Bronchodilatory, Fractions, Guinea pig, Nigella sativa, Trachea
Nigella sativa L. (Ranunculaceae) is a grassy plant
that grows in temperate and cold climate areas and
has green to blue flowers and small black seeds. The
seeds contain thymoquinone, monotropens such as
p-cymene and α-pinene1, nigellidine2, nigellimine3
and a saponin4. The chemical composition of the plant
was summarized in a recent review5.
The therapeutic use of the seeds of N. sativa for the
treatment of asthma and dyspene have been described
in ancient Iranian litrature6. The relaxant effects of the
volatile oil of seeds on different isolated smooth
muscle preparations like rabbit aorta7, rabbit
jejunum8, and guinea pig tracheal muscle9 have been
reported. Mahfouz and El-Dakhakhnsy10 reported that
the volatile oil from N. sativa protected guinea pigs
___________
Present addresses:
1
Deptartment of Physiology and Pharmacological Research Center
of Medicinal Plants, Medical School, Mashhad University of
Medical Sciences, Mashhad, 9177948564, Iran
Telephone: 0098 511 8828565
Fax: 0098 511 8828564
E-mail: [email protected]
2
Deptartment of Basic Sciences, Islamic Azad University,
Tabriz branch, Iran
against histamine-induced bronchospasm, but it did
not block histamine H1 receptors in isolated tissues.
Both systemic and local administrations of essential
oil from this plant have been shown to have antiinflammatory activity11. The therapeutic effect of
N. sativa oil on patients with allergic diseases
(allergic rhinitis, bronchial asthma, atopic eczema)
was also demonstrated12. In addition, Labib Salem5
reported the potent immunomodulatory effects of
seeds of the plant.
Different pharmacological effects of N. sativa on
guinea pig tracheal chains have been reported13-17. The
antitussive effect of this plant on guinea pig18 was
also demonstrated.
The objective of present study is to evaluate, the
effects of n-hexane, dichloromethane, methanol and
aqueous fractions of N. sativa on isolated tracheal
chains of guinea pigs.
Materials and Methods
Plant and fractions— Nigella sativa was collected
from Torbat Heydarieh (north east Iran) during spring
of 2002, and dried in shade at room temperature. The
plant was identified by botanists of Ferdowsi
806
INDIAN J EXP BIOL, DECEMBER 2008
University of Mashhad, and the voucher specimen
(293-0303-1) preserved. For separating fractions
300 ml n-hexane was added to 500 g of the chopped,
dried seeds and the solution was kept at the room
temperature for 48 hr. The solution was then decanted
and the solvent was evaporated. The dichloromethane
was added to the remaining powder and the mixture
was allowed to remain at the room temperature for
48 hr. The solution was decanted and the solvent was
evaporated. Methanol was added to the remaining
powder and the mixture was allowed to remain at the
room temperature for 48 hr. The solution was
decanted and evaporated. Finally, distilled water was
added to the remaining powder and the mixture was
allowed to remain at the room temperature for 48 hr
and the solution was decanted and solvent was
evaporated (Fig. 1). In these manners all of solvents
were added until the solutions became clear. Then the
final solution contained 40 g/100 ml extract (40 g%).
Tissue preparation ⎯ Adult Dunkin-Hartley
guinea pigs (400-700 g, both sexes) were used.
Animals were housed in big cages (4–5 per cage) with
free access to food and water ad libitum, maintained
at 22º ± 2ºC on a 12 hr light/dark cycle (light period
0700 and 1900 hrs). Guinea pigs were sacrificed by a
blow on the neck and tracheas were removed. Each
trachea was cut into 10 rings (each containing 2-3
cartilaginous rings). All the rings were then cut open
opposite the trachealis muscle, and sutured together to
form a tracheal chain19. Tissue was then suspended in
a 20 ml organ bath (schuler organ bath type 809,
March- Hugstetten, Germany) containing KrebsHenseliet solution containing (mM): NaCl 120,
NaHCO3 25, MgSO4 0.5, KH2PO4 1.2, KCl 4.72,
CaCl2 2.5 and dextrose 11. The Krebs solution was
maintained at 37oC and gassed with 95% O2 and 5%
CO2. Tissue was suspended under an isotonic tension
of 1 g and allowed to equilibrate for at least 1 hr and
washed with Krebs solution every 15 min.
Protocol of experiments ⎯ The tissue was
contracted with KCl (60 mM) or methacholine
(10 μM). The relaxant effects of four cumulative
concentrations (0.8, 1.2, 1.6 and 2.0 g/100 ml) of
n-hexane, dichloromethane, methanol and aqueous
fractions of N. sativa and theophylline anhydrous
(Sigma Chemical Ltd UK) (0.2, 0.4, 0.6 and 0.8 mM)
as positive control, and saline (1 ml) as negative
control were examined. To produce the first
concentration of each fraction, 0.4 ml of 40 g% was
added to a 20 ml organ bath. This will produce a
0.8 g% concentration of fraction in organ bath as
follows: 0.4 × 40 g% = 16 g%, 16 g%/20 ml = 0.8 g%
(0.8 g/ 100 ml). For other three concentrations, 0.2 ml
of 40 g% was added to organ bath respectively, three
times. For theophylline, 0.2 ml of 20 mM
concentrated solution was added to organ bath
4 times. The consecutive volumes were added to
organ bath at 5 min intervals.
In each experiment the effect of four cumulative
concentrations from each fraction, theophylline or
saline on KCl or methacholine contracted tracheal
smooth muscle was measured after exposing tissue to
each concentration of the solution for 5 min. A
decrease in tone was considered to be a relaxant
(bronchodilatory) effect and expressed as positive
percentage change in proportion to the maximum
contraction. An increase in tone was considered as a
contractile (bronchoconstrictory) effect which was
expressed as negative percentage change20.
The relaxant effect of different solutions was tested
with following two different experimental designs,
(n= 6 for each group):
1. On tracheal chains contracted by 60 mM KCl
(group 1 experiments).
2. On tracheal chains contracted by 10 µM
methacholine hydrochloride (Sigma Chemical Ltd
UK), (group 2 experiments).
Fig. 1—Separation of the different fractions from the seeds of
N. sativa
The relaxant effects in two groups of experiments
were examined in two different sets of tracheal
chains. All the experiments were performed randomly
with a 1 hr resting period of tracheal chains between
two experiments while washing the tissues every
BOSKABADY et al.: RELAXANT EFFECT OF FRACTIONS FROM NIGELLA SATIVA
807
(P<0.001 for all concentrations). Two last
concentrations (1.6 and 2.0 g%) of n-hexane fraction
and three last concentrations (1.2, 1.6 and 2.0 g%) of
dichloromethane and methanol fractions showed
small but significant contractile effects compared to
that of saline in this group (Table 1).
In group 2 experiments all concentrations of
theophylline and methanol fraction, three last
concentrations (1.2, 1.6 and 2.0 g%) of
dichloromethane and two higher concentrations
(1.6 and 2.0 g%) of n-hexane fractions showed
significant relaxant effects compared to that of saline.
However, aqueous fraction in both group 1 and group
2 did not show any significant relaxant effect on
tracheal chains (Table 2).
Comparison of the relaxant effect of theophylline
with different fractions ⎯ In group 1 the relaxant
effect of all concentrations and in group 2, the
relaxant effect of all concentrations of different
fractions except the first concentration of
dichloromethane and methanol fraction were
significantly less than those of theophylline
(Tables 1 and 2).
Comparison of the relaxant effect of different
fractions ⎯ In group 1, the effect of the higher
concentration of methanol and dichloromethane
15 min with Krebs solution. In all experiments
responses were amplified with amplifier (ML/118
quadribridge amp, March-Hugstetten, Germany) and
recorded on powerlab (ML-750 ,4 channel recorder,
March-Hugstetten, Germany). The research protocol
was approved by ethical committee of Mashhad
University of Medical Sciences and the norms of
international animal ethics were followed.
Statistical analysis ⎯ The data were expressed as
mean±SE. Data of relaxant effects of different
concentrations of each fraction were compared with the
results of negative and positive control using paired t
test. The data of relaxant effects obtained in two groups
of experiments were compared using unpaired ‘t’ test.
The relaxant effects of different concentrations of four
different fractions were compared with each other
using One-way Analysis of Variance (ANOVA) with
Tukey-Kramer post test. The relaxant effect of four
fractions and theophylline were related to the log
concentrations using least square regression.
Significance was accepted at P<0.05.
Results
Relaxant effect ⎯ In group 1 experiments all
concentrations of only theophylline showed
significant relaxant effects compared to that of saline
Table 1—Relaxant effect of four different fractions (n-hexane, dichloromethane, methanol and aqueous) from N. sativa in comparison
with negative control (saline) and positive control (theophylline) in group 1 experiments (contracted tracheal chains with 60 mM KCl)
[Values are mean± SE from 6 animal in each group]
Concentration
1
2
3
4
Saline
N-hexane Fraction
0.42±0.27
a,c
-2.67±1.6
-4.75±1.92 a,c
-8.12±2.53d,c
-12.41±3.03d,c
Dichlorom fraction
a,c
-2±1.63
-8.28±2.91d,c
-14.33±3.4a,c
-22±3.31b,c
Methanol fraction
a,c
-3.34±1.74
-9.33±3.28d,c
-16.17±3.47b,c
-21.34±3.78b,c
Aqueous fraction
a,c
-0.84±0.84
-2.91±2.08a,c
-4.59±2.91a,c
-1.25±4.27a,c
Theophylline
17.22±2.34 b
48.50±4.30b
66.69±3.43b
81.32±2.51b
The four different concentrations for fractions were 0.8, 1.2, 1.6 and 2.0 g%, and for theophylline, 0.2, 0.4. 0.6 and 0.8 mM.
P values: anon significant when compared with saline; b<0.001 when compared with saline; c<0.001 when compared with theophylline;
d
<0.05 when compared with saline; e<0.005 when compared with saline; f<0.005 when compared with theophylline; gnon significant
when compared with theophylline
Table 2—Relaxant effect of four different fractions (n-hexane, dichloromethane, methanol and aqueous)
from N. sativa in comparison with negative control (saline) and positive control (theophylline) in group 2
experiments (contracted tracheal chains by 10 μM methacholie)
[Values are mean± SE from 6 animal in each group]
Concentration
Saline
N-hexane
Fraction
Dichlorom
fraction
Methanol
fraction
Aqueous
fraction
Theophyline
1
2
3
4
0.55±0.37
0.83±0.83a,f
4.17±2.007a,f
8.34±2.79d,c
11.67±3.8d,c
5.84±2.71a,g
11.67±3.57d,c
17.5±4.95e,c
25±7.07b,c
8.34±2.47d,g
18.04±3.28b,f
27.48±4.73b,c
40.33±6.86b,f
0.00±0.00
1.87±1.2a,c
3.75±2.4a,c
10±4.65a,c
15.42±3.56e
43.75±4.99 b
62.50±5.28b
82.50±6.55b
For other details Table I.
INDIAN J EXP BIOL, DECEMBER 2008
808
fractions were significantly lower than that of aqueous
fraction (P<0.01 for both fraction), (Fig. 2).
In group 2 the relaxant effect of three last
concentrations of methanol fraction were significantly
greater than those of n-hexane fraction and aqueous
fractions (P<0.05 to <0.01), (Fig. 2).
Comparison of the relaxant effect between two
groups of experiments ⎯ The relaxant effect of
different
concentrations
of
methanol
and
dichloromethane fractions were significantly greater
in group 2 compared to group 1 experiments. The
relaxant effect of three last concentrations of n-hexane
Fig. 2—Concentration response curves of the relaxant effect of nhexane (a), dichloromethane (b), methanol (c), aqueous fractions
(d) from N. sativa and theophylline (e) in two groups of
experiments. group 1; KCl induced contraction of tracheal chains
(ο) and group 2; methacholine induced contraction of tracheal
chains ( ). Statistical differences in the relaxant effect of different
concentrations of each fraction between group 1 with those of
group 2; NS: non-significant difference. [P values: *<0.05;
**<0.01; ***<0.001. The concentration unit for fractions was g%,
and for theophylline, mM].
fraction were also significantly greater in group 2
compared to group 1 experiments. However, there
was no significant difference in the relaxant effect of
different concentrations of theophylline and aqueous
fraction between two groups (Fig. 2a and b).
Correlation between concentrations of solutions
and their relaxant effect ⎯ There were significant
positive correlations between the relaxant effects and
concentrations for all fractions (except aqueous
fraction) in group 2 and for theophylline in both
groups. However, there were significant negative
correlations between the relaxant effects and
concentrations for all fractions (except aqueous
fraction) in group 1 (Table 3).
Discussion
In the present study the relaxant (bronchodilatory)
effects of four different fractions (n-hexan,
dichloromethane, methanol and aqueous) from
N. sativa were studied in comparison with saline as
negative control and theophylline as positive control.
In group 1 experiments (contracted tracheal chains by
KCl) while theophylline showed significant relaxant
effect, different fraction of N. sativa did not show any
relaxant effect. However, in group 2 experiments
(contracted tracheal chains by methacholine) all
concentrations of theophylline and methanol fraction,
three last concentrations (1.2, 1.6 and 2.0 g%) of
dichloromethane and two higher concentrations
(1.6 and 2.0 g%) of n-hexane fractions showed
significant relaxant effects on tracheal smooth muscle.
The absence of relaxant effect of different fractions
from N. sativa in group 1 may indicate an opening
effect for fractions of this plant on potassium channels
because the bronchodilatory effect of potassium
channel opening has been demonstrated previously21.
If the fractions from N. sativa had a potassium
channel opening effect, they would not have a
relaxant effect on tracheal chains contracted by KCl,
while they could show a relaxant effect when the
tracheal chain was contracted by methacholine.
However, with regard to bronchodilatory effect of
calcium channel blockers21,22 and because KCl affect
Table 3—Correlation (r) between the relaxant effect s of four different fractions (n-hexane, dichloromethane, methanol and aqueous)
from N. sativa and theophylline with their log concentrations in two groups of experiments
Different
N-hexane fraction
Dichloromethane fraction
Methanol fraction
Aqueous fraction
Theophylline
solutions
Group 1
Group 2
R
-0.553
0.571
P value
<0.005
<0.005
r
-0.739
0.536
P value
<0.001
<0.005
r
-0.688
0.739
P value
<0.001
<0.001
r
-0.076
0.447
P value
NS
<0.05
r
0.957
0.899
P value
<0.001
<0.001
BOSKABADY et al.: RELAXANT EFFECT OF FRACTIONS FROM NIGELLA SATIVA
calcium channels23,24, the result of the present study
did not suggest an inhibitory effect of this plant on
calcium channels of guinea pig tracheal chain. In fact
these findings are supported by the results of previous
study16 for aqueous and macerated extracts from N.
sativa. The greater relaxant effect of N-hexane,
dichloromethane and methanol fractions in group 2
may indicate an inhibitory effect on muscarinic
receptors which is supported by the results of the
previous study for the extracts of the plant13.
As the mechanism of action of the fractions was
not known, a tracheal relaxant drug with non specific
activity (theophylline) was chosen. Although
mechanisms of action of methylxanthines remains
poorly understood, it is believed that these drugs
facilitate bronchodilation by inhibition of different
families of phosphodiesterases (PDE), leading to
increased cAMP, prostaglandin inhibition, adenosine
receptor blockade, enhancement of endogenous
catecholamine levels, inhibition of cGMP, and
enhancement of the translocation of intracellular
calcium25,28. In addition, methacholine also induces
contraction by binding to muscarinic subtype 3 (M3)
receptor activates a G protein and phospholipase C
(PLC) which converts phosphoinositol (PIP2) to IP3
and diacylglycerol (DAG). IP3 releases Ca2+ from
intracellular stores causing smooth muscle
contraction. This is inhibited by increase in cAMP
due to different drugs, which activate adenylyl
cyclase (AC) via a stimulatory G protein (Gs),
converting ATP to cAMP. cAMP is degraded by
phosphodiesterase (PDE) to 5’-AMP29. Therefore
another possible mechanism responsible for relaxant
effect of fractions of N. sativa is increasing cAMP by
PDE inhibition or other mechanism similar to action
of β-agonist drugs.
The relaxant effect of 1.2, 1.6 and 2.0 g%
concentrations of methanol fraction was significantly
greater than those of n-hexan and aqueous fractions.
The findings of the present study indicated that mainly
lipid soluble ingredients of N. sativa are responsible for
the relaxant effect of the seeds on tracheal chains of
guinea pigs. However, more studies are required for the
exact mechanism(s) and the effective substances
causing the relaxant effect of this plant.
In conclusion, the results of the present study
showed a potent relaxant effect of mainly lipid
fractions from N. sativa on tracheal chains of guinea
pigs that was lower than that of theophylline at
concentrations used. The most potent relaxant effect
809
was shown by methanol and dichloromethane
fractions.
Acknowledgement
This study was financially supported by the
Research Council of Mashhad University of Medical
Sciences.
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